Rapidly acting prourokinase

ABSTRACT

A human prourokinase-like polypeptide having the following amino acid sequence: 
     
         (Met).Ser.sup.1 -X.sup.156.Y.sup.157.Z.sup.158 -- 
    
     wherein Met is an occasionally present methionine, Ser is the first N-terminal serine, X is the 156th arginine or other amino acid, Y is the 157th proline, glycine, alanine or valine, Z is the 158th lysine or arginine, and the solid lines represent the same amino acid sequences as corresponding parts of an amino acid sequence of a natural type human prourokinase or a human prourokinase-like polypeptide wherein the 135th lysine is changed to an amino acid other than a basic amino acid, or substantially the same amino acid sequence as the above-mentioned amino acid sequence, is provided. 
     Moreover, a DNA segment coding for the above-mentioned polypeptide; a plasmid containing the DNA segment; E. coli transformed with the plasmid; a process for production of the polypeptide characterized by culturing the E. coli; a pharmaceutical preparation containing the polypeptide; use of the polypeptide for prophylaxis or treatment of thrombus formation; and use of the polypeptide for the production of pharmaceutical preparation used for that purpose.

TECHNICAL FIELD

The present invention relates to rapidly acting human-prourokinase-likepolypeptides, DNA segments coding for the polypeptide, plasmidscontaining the DNA segment, Escherichia coli containing the plasmid, aprocess for the production of human prourokinase-like polypeptides usingthe E. coli, and the use of the polypeptides.

BACKGROUND ART

Human urokinase is an enzyme found as a trace in human urine capable ofactivating inactive plasminogen into plasmin, and the plasmin thusformed is capable of dissolving fibrin. This urokinase consists of twopolypeptide chains linked together by a disulfide bond. On the contrary,human prourokinase is a single chain polypeptide in which the aforesaidtwo polypeptide chains are joined together through an amide bond.Although this prourokinase itself does not possess the aforementionedactivity, it can be converted to the preceding active urokinase bycutting one amide bond.

Japanese Patent Application No. 61-12984 (Japanese Unexamined PatentPublication No. 62-143686) (EP 0210279) discloses stabilized humanprourokinase-like polypeptides wherein the 135th amino acid and the157th amino acid are varied, but these are intended to inhibit acleavage between the 135th amino acid and the 136th amino acid, and/or acleavage between the 158th amino acid and the 159th amino acid, andtherefore, are completely different from the polypeptides of the presentinvention.

Both the prourokinase described in EP 0200451 and the prourokinase-likepolypeptides described in Japanese Patent Application No. 61-12984(Japanese Unexamined Patent Publication No. 62-143686) (EP 0210279) donot practically exhibit the activities thereof at a site at whichthrombus is present, and therefore it is assumed that, even thoughapplied in a large amount, side effects such as systemic hemorrhage areprevented. Nevertheless, it is considered that the conversion ratethereof to a two chain type is low.

As described above, the human urokinase is an activated enzyme, and theactivity thereof is rapidly lost due to the presence of a large amountof various inhibitors in blood, and therefore, where used as atherapeutic agent, a large amount thereof must be administered. As aresult, a side effect of a systemic formation of plasmin occurs, whichtends to cause systemic hemorrhage.

Although prourokinase, also called single-chain urokinase, is usuallyinactive in plasma, it exhibits a weak activity at a site at whichthrombus is present, to convert plasminogen to plasmin. It is consideredthat, since a small amount of plasmin produced by the action of a tissueplasminogen activator or prourokinase converts prourokinase to atwo-chain type high molecular weight urokinase having a high activity,the conversion of plasminogen to plasmin rapidly proceeds, and thethrombus is lyzed (literature 1).

Where prourokinase is used as thrombolytic agent, a small applicationamount does not produce a sufficient amount of plasmin, and therefore,the thrombus cannot be efficiently lyzed. Conversely, in the case of anexcess application amount, even though a large amount of plasmin istemporarily produced, an increased amount of the plasmin does notincrease the thrombolysis, due to the distance thereof from a thrombussite, resulting in a decreased thrombolysis efficiency. Moreover, aportion of plasmin thus produced is inactivated by inhibitors such as α₂-antiplasmin, and another portion activates prourokinase at a site atwhich thrombus is not present, resulting in side effects such assystemic hemorrhage. Therefore, when administering prourokinase, a doseand manner of administration which will not produce a temporal excessamount of plasmin is described. Moreover, it is reported thatprourokinase, when cleaved at a peptide bond between Arg 156 and Phe157, is no longer activated by plasmin (literature 2), and accordingly,the prourokinase cannot properly exhibit its function in the presence ofthrombin.

EP 0200451 describes protease-resistant urokinases wherein the 156th to158th amino acids are changed, but all of these urokinases are intendedonly to prevent protease cleavage at a site between the 158th amino acidand the 159th amino acid, on the assumption that the cleavage of thissite is not necessary when activating prourokinase, and accordingly, arecompletely different from the present polypeptides.

Accordingly, a new type of human prourokinase is sought which, whilemaintaining a high thrombus specificity of human prourokinase, does notexhibit side effects due to a large application amount, is rapidlyactivated during and after the formation of thrombus, and therefore,does not have the above-mentioned disadvantages of human urokinase,human prourokinase, and aforementioned derivatives thereof. Such a humanprourokinase is not known.

DISCLOSURE OF THE INVENTION

Accordingly, the present invention provides human prourokinase-likepolypeptides which have a higher specificity to thrombus in comparisonwith a natural type human prourokinase when administered to an organism,do not exhibit side effects when administered in a large amount, areactivated by thrombin, have an improved rapid action, and preferably aredifficult to inactivate by a cleavage at a site between the 156th aminoacid and the 157th amino acid by thrombin; a gene system for theproduction of the polypeptide and a process for production of thepolypeptide using the gene system; and the use of the polypeptide.

More specifically, the present invention provides a humanprourokinase-like polypeptide having the following amino acid sequence:

(Met)·Ser¹ -X¹⁵⁶ ·¹⁵⁷ ·Z¹⁵⁸

wherein Met is an occasionally present methionine, Ser is the firstN-terminal serine, X is the 156th arginine or other amino acid, Y is the157th proline, glycine, alanine or valine, Z is the 158th lysine orarginine, and the solid lines are the same amino acid sequences ascorresponding parts of an amino acid sequence of a natural type humanprourokinase or a human prourokinase-like polypeptide wherein the 135thlysine is changed to an amino acid other than a basic amino acid, or hassubstantially the same amino acid sequence as the above-mentioned aminoacid sequence.

The present invention also provides a DNA segment coding for theabove-mentioned human prourokinase-like polypeptide.

Moreover, the present invention provides a plasmid containing theabove-mentioned DNA segment, control regions for expression thereof, anda DNA sequence necessary for reproduction in E. coli.

Still further, the present invention provides E. coli transformed withthe plasmid.

The present invention also provides a process for the production ofhuman prourokinase-like polypeptide, characterized by culturing theabove-mentioned transformed E. coli and recovering the polypeptide fromthe culture broth.

The present invention further provides a pharmaceutical preparationcomprising the above-mentioned human prourokinase-like polypeptidetogether with a pharmaceutical excipient.

The present invention also encompasses the use of the above-mentionedhuman prourokinase-like polypeptide for the production of a thrombolyticpreparation.

The present invention also provides a prophylactic or therapeutic methodagainst thrombus formation, characterized by administering theabove-mentioned human prourolinase to a patient.

The present rapidly acting human prourokinase, not only are activated byplasimin, but also are rapidly activated by thrombin produced only at atime of thrombus formation, while natural type human prourokinase doesnot have the latter property. Therefore, when the presentprourokinase-like polypeptides are administered, an expression of theactivity thereof is limited to sites at which thrombus is present, incomparison to the conventional human prourokinase, and occurs rapidly atan early stage of the thrombus formation, and therefore, are promisingas a rapidly acting thrombolytic agent with less side effects such assystemic hemorrhage.

Further, where the gene containing a DNA segment coding for the presenthuman prourokinase-like polypeptide is used, the polypeptide isefficiently expressed, and therefore, the polypeptide can beeconomically produced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-1 to 1-4 represent a nucleotide sequence of cDNA coding for anatural type human prourokinase and a corresponding amino acid sequence;

FIGS. 2-1 and 2-2 represent the construction of an intermediate plasmidpMUT9Q from starting plasmids pMUT4L and pMUP1pm;

FIG. 3 represents the construction of plasmid pIO-1 from plasmid pMUT9Q;

FIG. 4-1 represents nucleotide sequences of synthetic DNA oligomerscoding for mutated amino acids at the 156th, 157th and 158th positionsin human prourokinase-like polypeptides of the present invention, andcorresponding amino acid sequences;

FIGS. 4-2 and 4-3 represent nucleotide sequences of further similarsynthetic DNA oligomers and corresponding amino acid sequences;

FIG. 5 represents a process for the construction of plasmid pMUT9Q-RPK;

FIG. 6 represents a process for the construction of plasmid pMUT9Q-RPR;

FIG. 7 represents a process for the construction of plasmid pMUT9Q-QPR;

FIG. 8 represents a process for the construction of

FIG. 9 represents an SDS-polyacrylamide gel electrophoresis pattern forpurified preparation of the present mutant prourokinases Q-RPK andQ-RPR;

FIGS. 10 to 11 represent the progress of activation of purifiedpreparation of the present mutant prourokinases Q-RPK and Q-RPR byplasmin or thrombin; and

FIG. 12 shows the result when the residual activity of purifiedpreparations of the present mutant prourokinases Q-RPK and Q-RPR afterthrombin treatment is plotted in relation to the thrombin treatmenttime.

Note, the results obtained when natural type prourokinase and a mutantprourokinase Q(135) D(157) are used as control preparation are alsoshown in FIGS. 9

BEST MODE OF CARRYING OUT THE INVENTION A. Rapidly acting humanprourokinase-like polypeptide

The present rapidly acting human prourekinase-like polypeptide has thefollowing amino acid sequence:

    (Met)·Ser.sup.1 -X.sup.156 ·Y.sup.157 ·Z.sup.158 --

wherein Met is an occasionally present methionine, Ser is the firstN-terminal serine, X is the 156th arginine or other amino acid, Y is the157th proline, glycine, alanine or valine, Z is the 158th lysine orarginine, and the solid lines are the same amino acid sequences ascorresponding parts of an amino acid sequence of a natural type humanprourokinase or a human prourokinase-like polypeptide wherein the 135thlysine is changed to an amino acid other than a basic amino acid, or hassubstantially the same amino acid sequence as the above-mentioned aminoacid sequence.

Human prourokinase is a polypeptide consisting of 411 amino acids, andthe amino acid sequence thereof is already known (literature 3). Theplasminogen activator activity of human prourokinase is low, and it isconsidered that the activity thereof is exhibited in vivo by cleavage ofa peptide bond between the 158th lysine and the 159th isoleucine(literature 3), usually by plasmin. In a normal organism, plasmin ispresent as plasminogen, which is an inactive precursor, but oncethrombus has been formed by a pathogenic cause, the vascular wall andthe like is stimulated to secrete the tissue plasminogen activator whichthen generates plasmin on a thrombus surface, and the plasmin triggersthe onset of thrombolysis (literature 1). Therefore, when prourokinaseis administered to an organism, an expression of the activity thereof islimited to a site at which plasmin is present, i.e., thrombus surface,resulting in a prevention of the tendency to systemic hemorrhage foundwhen human urokinase is administered. Where prourokinase is used asthrombolytic agent, however, a small application amount does not producea sufficient amount of plasmin, and therefore, the thrombus cannot beefficiently lyzed, but in the case of an excess application amount, eventhough a large amount of plasmin is temporarily produced, an increasedamount of the plasmin does not increase the thrombolysis due to thedistance thereof from a thrombus site, resulting in a decreasedthrombolysis efficiency. Moreover, a portion of plasmin thus produced isinactivated by inhibitors such as α2-antiplasmin, and another portionactivates prourokinase at a site at which thrombus is not present,resulting in side effects such systemic hemorrhage. Therefore, in theadministration of prourokinase, the dose and manner of administrationwhich do not produce a temporarily excess amount of plasmin is desired.Moreover, it is reported that prourokinase, when cleaved at a peptidebond between Arg 156 and Phe 157, is no longer activated by plasmin(literature 2).

Thrombin converts fibrinogen to fibrin monomer at a final stage in ablood coagulation cascade, and the fibrin monomers self-associate toform a large fibrin network which forms thrombus. On the other hand,although a portion of the thrombin is captured in the fibrin networkduring the above-mentioned process, another portion of the thrombin isreleased to the thrombus surface, and acts to expand the network.

After the formation of thrombus, although the thrombus which escapedcapture is rapidly inactivated by antithrombin III or the like, thecaptured thrombin is diffused and appears on the thrombus surface(literatures 4 and 5) while maintaining the activity thereof. Moreover,since the captured thrombin is released when the thrombus is dissolvedby fibrolytic enzyme (literature 4), a reformation of the thrombusoccurs.

The present inventors created by replacement of the 157th phenylalaninewith proline, completely novel prourekinase having the followingproperties:

i) the rate of activation by plasmin is lower than that of natural typeprourokinase; and

ii) it is activated by thrombin, in contrast with natural typeprourokinase which is not activated by thrombin.

Due to the property i), the present prourokinase is expected not toexhibit side effects such as systemic hemorrhage even if administered ina large amount. Moreover, taking into account the above-mentionedlocalization of thrombin, due to the property ii), it is expected thatthe present prourokinase will not only rapidly andthrombus-site-specifically exhibit the activity thereof from an earlystage of thrombus formation to termination thereof, but also willexhibits an effective lytic activity when thrombus is reformed bythrombin released after thrombolysis.

Note, the above-mentioned facts originally found by the presentinventors relating to prourokinase are reasonably explained according tothe substrate specificity of thrombin (literature 6). Namely, in thefollowing amino acid sequence: ##STR1## wherein P₂, P₁ and P₁, representany amino acid, and the symbol ↓ represents a cleavage site forthrombin, where P₁ is lysine or arginine, and P₂ is proline, glycine,alanine or valine, thrombin cleaves a peptide bond between P₁ and P₁ ',with a proviso that when P₁, is proline the cleavage does not occur.Accordingly, the response of natural type prourokinase to thrombin wherethe 157th amino acid is phenylalanine and of a prourokinase wherein the157th amino acid has been replaced with proline, is summarized asfollows: ##STR2##

Namely, for natural type prourokinase, first in a sequence of the 155thproline, the 156th arginine and the 157th phenylalanine, a peptide bondbetween the 156th arginine and the 157th phenyl alanine is easilycleaved by thrombin, and therefore, the natural type prourokinase isinactivated, while in a sequence of the 157th phenylalanine, the 158thlysine and the 159th isoleucine, it is difficult for thrombin to cleavea peptide bond between the 158th lysine and the 159th isoleucine, andtherefore the natural type prourokinase is not activated by thrombin Onthe other hand, for the present prourokinase wherein the 157th aminoacid is proline, first in a sequence of the 155th proline, the 156tharginine and the 157th proline, a peptide bond between the 156tharginine and the 157th proline is not cleaved by thrombin, andtherefore, the present prourokinase is not inactivated, while in asequence of the 157th proline, the 158th lysine and the 159thisoleucine, a peptide bond between the 158th lysine and the 159thisoleucine is easily cleaved by thrombin, and therefore, the presentprourokinase is activated.

Further, the reason why the prourokinase, wherein the 157th amino acidhas been replaced with proline, was activated also by plasmin can beexplained as follows. Since the 157th site is easily affected by plasminbecause this site is originally a cleavage site for plasmin, the plasmincleavage action is not largely affected by the replacement of the 157thamino acid.

According to the present invention, taking an account theabove-mentioned substrate specificity of thrombin, as embodiments ofcombinations of the above-mentioned X, Y and Z, the followingpolypeptides were prepared.

    ______________________________________                                        Symbol        X         Y         Z                                           ______________________________________                                        (1) RPK       Arg       Pro       Lys                                         (2) RPR       Arg       Pro       Arg                                         (3) QPR       Gln       Pro       Arg                                         (4) SGR       Ser       Gly       Arg                                         ______________________________________                                    

As described hereinafter in detail, the effect of plasmin and thrombinon the activation of the abovementioned various polypeptides was tested,and as a result, all the above-mentioned four combinations of thereplacements were confirmed to provide these novel prourokinases withdesired properties, i.e., the property of being activated by plasmin andthrombin as well as the property of not being inactivated by thrombin.Moreover, as described hereinafter in detail, the effect of thrombin onthe lysis time of a fibrin clot was determined, and as a result, all theabove-mentioned four combinations of the replacements were confirmed toprovide the prourokinases with the desired property; i.e., the higherthe thrombin concentration, the faster the fibrin clot is dissolved.Accordingly, in view of the above it can be reasonably expected thatpolypeptides defined by all the combination of the definitions of X, Yand Z have the following properties; they are activated by plasmin andthrombin; they are not inactivated by thrombin; and the higher thethrombin concentration, the faster they dissolve the fibrin clot.Therefore, all the prourokinase-like polypeptides defined by theabove-mentioned combinations of the definitions X, Y and Z are withinthe scope of the present invention.

As combinations of X, Y and Z of the present invention, in addition tothe above-mentioned combinations (1) RPK, (2) RPR, (3) QPR, and (4) SGR,for example, the following combinations can be used:

    ______________________________________                                        (5) X-Gly-Lys  (TUK-XGK)                                                      (6) X-Ala-Lys  (TUK-XAK)                                                      (7) X-Ala-Arg  (TUK-XAR)                                                      (8) X-Val-Lys  (TUK-XVK)                                                      (9) X-Val-Arg  (TUK-XVR)                                                      ______________________________________                                    

wherein X represents Arg present in a natural polypeptide or any otheramino acid.

In the above-mentioned general formula, amino acid sequences representedby solid lines are the same as corresponding parts of an amino acidsequence of natural type human prourokinase or of human prouro-kinase-like polypeptides wherein the 135th lysine has been replaced byan amino acid other than a basic amino acid. As an amino acid sequenceof the natural type human prourokinase, there can be mentioned an aminoacid sequence consisting of 411 amino acids encoded by cDNAcorresponding to mRNA derived from a human kidney This amino acidsequence is set forth in FIGS. 1-1 to 1-3 by amino acid symbols composedof three capitalized alphabetical characters.

The human prourokinase-like polypeptide wherein the 135th lysine hasbeen replaced by an amino acid other than the basic amino acid isdescribed in detail in Japanese Patent Application No. 61-12984(Japanese Unexamined Patent Publication No. 62-143686) (EP 0210279). Asamino acids other than the basic amino acid for the 135th position,alanine, asparagine, aspartic acid, glutamine, glutamic acid,phenylalanine, glycine, isoleucine, leucine, methionine, serine,threonine, valine, tryptophan, tyrosine, proline and the like can bementioned

In the above-mentioned general formula, (Met) represents methionineoccasionally present adjacent to the N-terminal first Ser ofprourokinase.

The rapidly acting human prourokinase-like polypeptide of the presentinvention, in addition to the polypeptides comprising theabove-mentioned amino acid sequences, includes polypeptides havingessentially the same amino acid sequence as disclosed above. The term,essentially the same amino acid sequence, herein means those amino acidsequences wherein one or a few amino acids other than X, Y and Z in theabove-mentioned amino acid sequence are replaced by other amino acid(s)or deleted, or one or a few amino acids are added to the above-mentionedamino acid sequence, but the physiological properties of prourokinaseand the properties characteristic to the present invention are stillmaintained; i.e., easily activated by plasmin and thrombin. It is wellknown to a person with ordinary skill in the art that, in some cases,the change of an amino acid sequence in a peptide having a particularphysiological activity in a region not relating to the physiologicalactivity does not affect the physiological activity. Therefore,polypeptides containing the above-mentioned changes are within the scopeof the present invention so long as they have the characteristics of thepresent invention.

B. Genes system for human prourokinase and process for productionthereof

The present invention also relates to a gene system useful for theproduction of rapidly acting human prourokinase and a process for theproduction of the human prourokinase using that gene system. The genesystem herein includes DNA segments coding for a desired humanprourokinase-like polypeptide, expression plasmids containing the DNAsegment, and a host to which the expression plasmid has been introduced.

A DNA segment coding for a rapidly acting human prourokinase of thepresent invention is derived from a DNA coding for a natural humanprourokinase, or a DNA coding for a stabilized human prourokinase-likepolypeptide described in Japanese Patent Application No. 61-12984 (EP0210279). Namely, in these DNA's, codons coding for amino acidscorresponding to the target amino acids (the above-mentioned X, Y and Z)are mutated to convert these codons to codons coding for desired aminoacids. The mutation is introduced by replacing a DNA fragment containinga codon(s) coding for the target amino acid(s) with a synthetic DNAfragment wherein a coden(s) coding for the target amino acid(s) ischanged to a codon(s) coding for the desired amino acid(s). The DNAfragment to be replaced is preferably a relatively small fragmentgenerated by appropriate restriction enzyme(s). For the synthetic DNAfragment, as codons for the target amino acids and other amino acids,all codons in the degeneration, preferably codons easily expressed in ahost, are used and are selected so that a sequence of the codons doesnot form a folding structure at the mRNA level. Alternatively, wellknown M13 phages can be used to introduce the mutation.

Note, in an embodiment of the present invention, as a gene source codingfor prourokinase, plasmid pMUT4L and plasmid pMUPlpm are used. Theprocess for the production and characterization of these plasmids isdescribed in detail in Japanese Patent Application No. 61-12984(Japanese Unexamined Patent Publication No. 62-143686) (EP 0210279).Although the plasmid pMUT4L contains a DNA fragment coding for aminoacids of a natural prourokinase, for improvement of the expression, inthe N-terminal coding region thereof, the following codons are used inplace of codons in native cDNA. ##STR3##

Note, a process for the construction of pMUT4L is set forth in ReferenceExamples 1 and 2.

A DNA segment coding for the present rapidly acting human prourokinaseis recombinated to form an expression plasmid. The expression plasmid isintroduced into host cells to accumulate a desired human prourokinase inthe host cells or a culture broth. A process for the production of humanprourokinase, described in Japanese Patent Application No. 61-12984(Japanese Unexamined Patent Publication No. 62-143686) (EP 0210279)totally applies to the production of the rapidly acting humanprourokinases of the present invention. Although a definite embodimentof the process is set forth herein in the Examples, hosts and expressionvectors other than those described to the Examples also are within thescope of the present invention.

Where E. coli cells are used as host cells, the producedprourokinase-like polypeptide is usually recovered as an insolublepellet after disruption of the cells by ultrasonication or a Gaulinhomogenizer. After the pellet is dissolved in 4 M guanidinehydrochloride aqueous solution, a steric structure of the desiredproduct is restored by a reaction thereof with a thiol compound or anoxidation thereof in air in the presence of guanidine hydrochloride.Next, after recovery of the desired product by salting out, using, forexample, ammonium sulfate, the product is purified by chromatographyusing a hydrophobic interaction in the presence of guanidinehydrochloride or chromatography using an interaction with metal ions,although other conventional biochemical separation techniques can beused.

The present human prourokinase-like polypeptides are useful as aprophylactic or therapeutic agent, especially a therapeutic agent usedagainst the thrombus formation, and accordingly, are administeredparentally, for example, intravenously, intraperitoneally,subcutaneously or intramuscularly. The application dosage depends on thecondition of a patient and manner of application and the like.Pharmaceutical preparations for parenteral application are usually inthe form of a solution in a conventional injectable excipient, or alyophilized preparation obtained from such a solution.

Next, the present invention will be further definitely illustrated by,but is by no means limited to, the following Examples and ReferenceExamples

Note, the reaction conditions used in the Examples are as follows.

Reaction of each restriction enzyme

10 units of a restriction enzyme was added to 50 μl of the followingreaction mixture containing 1 μg of DNA (plasmid or DNA fragment), andthe mixture was incubated at the following temperatures. Where partialdigestion was carried out, 1 to 2 units of a restriction enzyme wereadded, and the incubation time was 0.5 to 1 hour.

    __________________________________________________________________________    Restriction                   Reaction                                        enzyme    Composition of reaction mixture                                                                   temperature                                     __________________________________________________________________________              10 mM Tris-HCl (pH 7.5),                                            Aat II    50 mM KCl, 10 mM MgCl.sub.2,                                                                      37° C.                                             1 mM dithiothreitol                                                            6 mM Tris-HCl (pH 7.9),                                            BamH I                        30° C.                                             150 mM NaCl, 6 mM MgCl.sub.2                                                  6 mM Tris-HCl (pH 7.4),                                             Ban II    50 mM NaCl, 6 mM MgCl.sub.2,                                                                      37° C.                                             10 mM β-mercaptoethanol                                                  10 mM Tris-HCl (pH 8.0),                                            Dra II    40 mM KCl, 7 mM MgCl.sub.2, 7 mM                                                                  37° C.                                             β-mercaptoethanol                                                         100 mM Tris-HCl (pH 7.5),                                          EcoR I                        37° C.                                             50 mM NaCl, 5 mM (MgCl.sub.2)                                                  10 mM Tris-HCl (pH 7.5),                                           Hind III                      37° C.                                             60 mM NaCl, 7 mM MgCl.sub.2                                                    6 mM Tris-HCl (pH 7.4),                                            Nar I                         37° C.                                             6 mM MgCl.sub.2, 6 mM β-mercaptoethanol                                   10 mM Tris-HCl (pH 7.5),                                           Pst I                         37° C.                                             100 mM NaCl, 10 mM MgCl.sub.2                                                 6 mM Tris-HCl (pH 7.4),                                             Sca I     100 mM NaCl, 6 mM MgCl.sub.2,                                                                     37° C.                                             1 mM dithiothreitol                                                           6 mM Tris-HCl (pH 8.0)                                              Sma I     20 mM KCl, 6 mM MgCl.sub.2, 6 mM                                                                  25° C.                                             β-mercaptoethanol                                              __________________________________________________________________________

Reaction for Blunt End Formation of DNA by T₄ DNA Polymerase

A 0.5 to 1 unit of T₄ DNA polymerase was added to 50 μl of the followingreaction mixture containing 1 to 2μg of a linear DNA, and the mixturewas incubated at 37° C. for one hour.

Composition of reaction mixture

67 mM Tris-HCl (pH 8.8), 6.7 mM MgCl₂, 16.6 mM (NH₄)₂ SO₄, 10 mMβ-mercaptoethanol, 6.7 μM ethylenediaminetetraacetic acid, 0.0167%bovine serum albumin, 330 μM dCTP, 330 μM dATP, 330 μM dGTP and 330 μMdTTP.

Reaction for Blunt End Formation of DNA by Klenow Fragment

A 0.5 to 1 unit of Klenow fragment was added to 50 μl of the followingreaction mixture containing 1 to 2 μg of a linear DNA, and the mixturewas incubated at 25° C. for one hour.

Reaction mixture

67 mM potassium phosphate buffer (pH 7.4), 6.7 mM MgC12 , 1 mMβ-mercaptoethanol, 33 μM dATP, 33 μM 7 dTTP, 33 μM dGTP and 33 μM dCTP.

Reaction for Joining DNAs by T₄ DNA Ligase

To 7.5 μl of a DNA solution containing DNA fragments (about 0.1 μg) tobe joined, were added 60 μl of A solution of "DNA ligation kit" fromTakara Shuzo K.K. and 7.5 μl of B solution containing T4 DNA ligase, andthe whole was mixed and incubated at 16° C. for 30 minutes.

Example 1 Construction of Expression Plasmid FIGS. 2-1 and 2-2

(FIGS. 2-1 and 2-2)

Plasmid pMUT4L having two pairs of tac promoter/ operator for expressionof prourokinase was digested with restriction endonucleases AatII andSmaI, and a DNA fraction of about 5.5 kb was isolated by electroelution.On the other hand, the plasmid pMUT4L was digested with restrictionendonucleases AatII and ScaI, and a DNA fragment of about 60 bp wasisolated by electroelution. These two DNA fragments were ligated usingT₄ DNA ligase, and E. coli was transformed. The transformant wasanalyzed by a rapid isolation method according to an alkaline lysismethod, and plasmid pMUT8L having a pair of a tac promoter/operator wasobtained.

Next, the pMUT8L was digested with restriction endonuclease HindIII,blunt-ended using a Klenow fragment, and after ligation with acommercially available PstI linker, digested with PstI. From theresulting digestion product, a 3.3 Kb DNA fragment was isolated byelectroelution. On the other hand, plasmid pMUPlpm having a PL promoterand a gene for prourokinase wherein the 135th lysine has been convertedto glutamine, was digested with a restriction enzyme PstI, and 1.2 kbDNA fragment was isolated by electroelution. These two DNA fragmentswere ligated by T₄ DNA ligase to obtain an expression plasmid pMUT9Lpmlhaving a gene for prourokinase wherein the 135th lysine has beenconverted to glutamine, downstream of the tac promoter/operator.

Next, this plasmid pMUT9Lpml was partially digested with a restrictionenzyme EcoRI, blunt-ended by a Klenow fragment, and self-circularizedusing a T₄ DNA ligase. From the resulting clones, a plasmid wherein onlythe EcoRI site present between a promoter/operator region and an S/Dregion has been converted to an XmnI site was selected, and designatedpMUT9Xpml.

Next, the pMUT9Xpml was partially digested with restriction enzymesBamHI and NarI, blunt-ended by a Klenow fragment, and self-circularizedusing a T₄ DNA ligase. From the resulting clones, a plasmid lacking aDNA fragment of about 200 bp between the BamHI site and NarI site,present upstream of a promoter/operator region, was selected anddesignated pMUT9Q. The pMUT9Q is an expression plasmid having a DNAfragment coding for prourokinase Q(135) wherein the 135th lysine hasbeen converted to glutamic acid, downstream of the tacpromoter/operator. Note, the mutant prourokinase Q(135) is the same as amutant prourokinase obtained from pMUP1pm.

Escherichia coli containing plasmid pMUP1pm used as a starting materialin this Example was deposited with the Fermentation Research Institute,Agency of Industrial Science and Technology as FERM BP-969 on Jan. 11,1985, as an international deposition under the Budapest Treaty.

Note, although as described above the present human prourokinase-likepolypeptide encoded in the plasmid pMUT9Q has glutamine as the 135thamino acid, a plasmid corresponding to the plasmid pMUT9Q but coding forthreonine as the 135th amino acid can be obtained in the same manner asdescribed above by using, in place of the above-mentioned plasmidpMUPlpm, a plasmid pMUT4Lpm2 containing a DNA fragment coding for ahuman prouro- kinase-like polypeptide having threonine as the 135thamino acid.

Escherichia coli χ1776/pMUT4Lpm2 containing the above-mentioned plasmidpMUT4Lpm2 was deposited as FERM BP-970 on Apr. 18, 1985 as aninternational deposition.

Similarly, by using a plasmid pMUT4Lpm3 in place of the plasmid pMUPlpm,a plasmid corresponding to the above-mentioned plasmid pMUT9Q but codingfor lysine as the 135th amino acid can be obtained Escherichia coliX1776/pMUT4Lpm3 containing the plasmid pMUT4Lpm3 was deposited as FERMBP-971 on Jul. 11, 1985, as an international deposition.

Example 2 Construction of Plasmid pIQ-Δ for Introduction of Mutation(FIG. 3)

The plasmid pMUT9Q obtained in Example 1 was completely digested with anrestriction enzyme Eco47III, and then partially digested with arestriction enzyme PstI. Next, after blunt end formation by T₄ DNApolymerase, the fragment was self-circularized using a T₄ DNA ligase.Among the resulting clones, a plasmid lacking only a Eco47III-PstIfragment of 0.77 kb was selected, and designated pIQ-Δ.

Similarly, a plasmid corresponding to the pIQ-Δ but coding for threonineor lysine as the 135th amino acid also can be obtained.

Example 3.

Construction of Q-RPK Type Mutant FIG. 5)

The plasmid pIQ-Δ obtained in Example 2 was digested with DraII andEcoRI, and a 3.5 kb DNA fragment was isolated by electroelution. On theother hand, two oligonucleotides, TUK-RPK (A) and TUK-RPK (B) (FIG.4-1), synthesized by a phosphite method were annealed, and ligated withthe 3.5 kb DNA fragment using T₄ DNA ligase to obtain an intermediateplasmid pIQ-RPK. Next, the pIQ-RPK was digested with a restrictionenzyme BanII, and a 0.54 kb DNA fragment was isolated by electroelution.On the other hand, the plasmid pMUT9Q obtained in Example 1 was digestedwith a restriction enzyme BanII, and a 3.8 kb DNA fragment was isolatedby electroelution. These two DNA fragments were ligated using a T₄ DNAligase to obtain a plasmid pMUT9Q-RPK. This plasmid is an expressionplasmid containing a DNA sequence coding for a mutant type humanprourokinase Q-RPK wherein the 157th phenylalanine has been converted toproline and the 135th lysine has been converted to glutamine, downstreamof the tac promoter/operator.

Escherichia coli JM103 (pMUT9Q-RPK) containing this plasmid wasdeposited with Deutsche Sammulung von Microorganismen as DSM 4187, onJul. 22, 1987 as an international deposition.

Example 4

Construction of Q-RPR Type Mutant (FIG. 6)

The plasmid pIQ-Δ obtained in Example 2 was digested with restrictionenzymes DraII and EcoRI, and a 3.5 kb DNA fragment was isolated byelectroelution. 0n the other hand, two oligomer TUK-RPR (A) and TUK-RPR(B) (FIG. 4-1) synthesized by a phosphite method were annealed andligated with the 3.5 kb DNA fragment using a T₄ DNA ligase, to obtain anintermediate plasmid pIQ-RPR. Next, the pIQ-RPK was digested with arestriction enzyme BanII, and a 0.54 kb DNA fragment was isolated byelectroelution. On the other hand, the plasmid pMUT9Q obtained inExample 1 was digested with a restriction enzyme BanII, and a 3.8 kb DNAfragment was isolated by electroelution. These two DNA fragments wereligated using T₄ DNA ligase to obtain a plasmid pMUT9Q-RPR This plasmidis an expression plasmid containing a DNA sequence coding for a mutanttype human prourokinase Q-RPR wherein the 157th phenylalamine has beenconverted to proline, the 158th lysine has been converted to arginine,and the 135th lysine has been converted to glutamine, downstream of thetac promoter/ operator.

Escherichia coli JM103 (pMUT9Q-RPR) containing this plasmid wasdeposited with Deutsche Sammulung von Microorganismen as DSM 4186 onJul. 22, 1987 as an international deposition.

Example 5 Construction of Q-QPR Type Mutant (FIG. 7)

The plasmid pIQ-Δ obtained in Example was digested with DraII and EcoRI,and a 3.5 kb DNA fragment was isolated by electroelution. On the otherhand, two oligonucleotides, TUK-QPR (A) and TUK-QPR (B) (FIG. 4-1),synthesized by a phosphite method were annealed, and ligated with the3.5 kb DNA fragment using T₄ DNA ligase to obtain an intermediateplasmid pIQ-QPR. Next, the pIQ-QPR was digested with a restrictionenzyme BanII, and a 0.54 kb DNA fragment was isolated by electroelution.On the other hand, the plasmid pMUT9Q obtained in Example 1 was digestedwith a restriction enzyme BanII, and a 3.8 kb DNA fragment was isolatedby electroelution. These two DNA fragments were ligated using a T₄ DNAligase to obtain a plasmid pMUT9Q-QPR. This plasmid is an expressionplasmid containing a DNA sequence coding for a mutant type humanprourokinase Q-RPR wherein the 156th arginine has been converted toglutamine, the 157th phenylalanine has been converted to proline, the158th lysine has been converted to arginine and the 135th lysine hasbeen converted to glutamine, downstream of the tac promoter/operator.

Escherichia coli JMI03 (pMUT9Q-QPR) containing this plasmid wasdeposited with Deutsche Sammulung von Microorganismen as DSM 4188 onJul. 22, 1987 as an international deposition.

Example 6 Construction of Q-SGR Type Mutant (FIG. 8)

The plasmid pIQ-I obtained in Example 2 was digested with restrictionenzymes DraII and EcoRI, and a 3.5 kb DNA fragment was isolated byelectroelution. 0n the other hand, two oligomer TUK-SGR (A) and TUK-SGR(B) (FIG. 4-1) synthesized by a phosphite method were annealed andligated with the 3.5 kb DNA fragment using a T₄ DNA ligase to obtain anintermediate plasmid pIQ-SGR.

Next, the pIQ-SGR was digested with a restriction enzyme BanII, and a0.54 kb DNA fragment was isolated by electroelution.

On the other hand, the plasmid pMUT9Q obtained in Example 1 was digestedwith a restriction enzyme BanII, and a 3.8 kb DNA fragment was isolatedby electroelution. These two DNA fragments were ligated using a T₄ DNAligase to obtain a plasmid pMUT9Q-SGR. This plasmid is an expressionplasmid containing a DNA sequence coding for a mutant type humanprourokinase Q-SGR wherein the 157th phenylalanine has been converted toproline, the 158th lysine has been converted arginine, and the 135thlysine has been converted to glutamine, downstream of the tacpromoter/operator.

Escherichia coli JM103 (pMUT9Q-SGR) containing this plasmid wasdeposited with Deutsche Sammulung von Microorganismen as DSM 4189 onJuly 22, 1987 as an international deposition.

Note, by using the same procedure as described in Examples 3 to 6,except for using a plasmid corresponding to the plasmid pIQ-I but codingfor threonine or lysine as the 135th amino acid, plasmids coding for thepresent polypeptide wherein the 135th amino acid is threonine or lysinecan be obtained.

Moreover, by using the same procedure as described above but usingsynthetic oligomers as set forth in FIG. 4-2, other mutants, forexample, TUK-XGK, TUK-XAK, TUK-XAR, TUK-KVK and TUK-XVR, can beproduced.

Example 7 Expression of Mutant Prourokinase Gene by E. coli

Plasmids pMUT9Q, pMUT9Q-RPK, pMUT9Q-RPR, and pMUT9Q-QPR obtained inExamples 1, 3, 4, and 5, respectively, were used to transform E. coliJM103 by a conventional procedure, and the resulting transformants werecultured in 5 ml of L-broth at 37° C. When the absorbance at 600 nmreached about 0.4 0.D, 50 μl of 100 mM isopropylthiogalactopyranoside(IPTG) was added, and culturing was continued for a further 4 hours toexpress each mutant prourokinase gene.

Example 8 Extraction of Gene Product from E. coli

Each culture broth obtained in Example 7 was centrifuged to recovercells corresponding to an amount of 7 0.D. ml, and these cells were thendisrupted in 1.4 ml of 50 ml Tris-HCl (pH 8.0) buffer containing 0.1 Msodium chloride by ultrasonication to a turbidity extent of not morethan 1 0.D at 600 nm, 0.8 ml of this disruptant solution was thencentrifuged at 15 Krpm for 5 minutes, and the supernatant was discarded.The precipitate was suspended in 0.16 ml of a 50 mM Tris-HCl buffercontaining 4 M guanidine hydrochloride, and the mixture was allowed tostand at a room temperature for 90 minutes to dissolve the precipitate.Next, 0.48 ml of a 50 mM Tris-HCl (pH 8.0) buffer containing 0.27 mMreduced glutathione and 1.33 mM EDTA was added, and the mixture wasallowed to stand at 25° C. for 15 hours. Then solid ammonium sulfate wasdissolved to 60% saturation to salt out a desired gene product, and tothe salting out product was added an amount of Tris-HCl (pH 8.0) buffercontaining 0.5 M sodium chloride, to prepare a crude extract of the geneproduct.

Example 9. Properties of Gene Product Extracted from E. coli (1) i)Measurement of urokinase activity by fibrin Plate Method

To 50 mM phosphate buffer (pH 7.4) containing 1% bovine fibrinogen,0.25% agarose and 0.1 M sodium salt was added bovine thrombin to a finalconcentration of one unit/ml, to prepare a fibrin plate, on which 10 tlof a sample (prepared by appropriately diluting the crude extractprepared in Example 6) was then spotted, and after incubation at 37° C.for 16 hours, a diameter of a holo was measured and compared with thatof a standard urokinase (Nippon Soda K.K.), to determine the activity.As a result, it was found that the urokinase activities of the crudeextracts obtained in Example 8 were 370 IU/ml for a product frompMUTQ-RPK (Q-RPK), 380 IU/ml for a product from pMUT9Q-RPR (Q-RPR), and360 IU/ml for a product from pMUT9Q-QPR (Q-QPR).

ii) Activation of Mutant Prourokinase by Plasmin

To 10 tl of a crude extract containing the mutant prourokinase Q-RPK,Q-RPR or Q-QPR was added 85 tl of 50 mM Tris-HCl (pH 8.0) containing 0.1M NaCl and 0.01% Triton X-100, then to this mixture was added 5 tl of anaqueous solution of human plasmin (specific activity: 15 casein units/mgprotein) to a final concentration of 0.1, 1, 10 or 50 tg/ml, andreaction was carried out at 37° C. for 15 minutes. The reaction wasterminated by adding 5 tl of an aqueous solution containing hirudine inan amount of two fold that of thrombin (ratio of activities).

iv) Measurement of Activity of Activated Mutant Prourokinases usingSynthetic Substrate S-2444

To 105 tl of Reaction Mixture Obtained in ii) and iii) was added 0.7 mlof 50 mM Tris-HCl (pH 8.0) buffer containing 0.2 mM S2444 (DaiichiKagaku Yakuhin), 0.1 M sodium chloride and 0.1% Triton X-100, andreaction was carried out at 37° C for 30 minutes. The reaction wasterminated by adding 0.1 ml of glacial acetic acid. Then an increase ofan absorbance at 405 nm for 30 minutes of the reaction was measured, andan activity of the sample was calculated from a value for a standardurokinase (Nippon Soda K.K.) (increase of absorbance at 405 nm for 30minutes is 0.0608 for one unit). The results are shown in Table 1, whichshows that the mutant prourokinase Q-RPK, Q-RPR, and Q-QPR are activatedby both plasmin and thrombin.

                  TABLE 1                                                         ______________________________________                                        Activation of Various Mutant Prourokinase                                     Mutant   Plasmin (μg/ml)                                                                             Thrombin (μg/ml)                                 prourokinase                                                                           0.1     1      10   50   0.1  1     10                               ______________________________________                                               (IU/ml)        (IU/ml)                                                 Q-RPK    30.5    154    284  315  4.2  28.4  166                              Q-RPR    15.8    121    272  315  75.8 385   428                              Q-QPR    24.2    151    305  385  82.1 369   390                              Q (135)  85.7    374    430  412  8.4  4.6    0                               ______________________________________                                    

Example 10 Property of Gene Product Extracted from E. coli (2)(measurement of Lysis Time for Fibrin Clot

First 0.2 ml of 50 mM phosphate buffer (pH 7.4) containing 15 mg/mlfibrinogen and 0.1 M sodium chloride was put into well of a 96-wellmicrotiter plate, and after adding 0.03 ml of an aqueous solutioncontaining 5 to 20 units/ml thrombin and 0.01 ml of the crude extract ofExample 6 containing 165 IU/ml of mutant prourokinase, 0.06 ml of 50 mMphosphate buffer (pH 7.4) heated to 65° C. containing 0.75% agarose and0.1 M sodium chloride was added and rapidly mixed (the time point was"0") and the mixture was incubated at 37° C. Although the mixtureimmediately gellated, it became transparent with an elapse of time. Thetime at which the gel started to become transparent and the time atwhich the gel became completely transparent were determined, and anintermediate time thereof was defined as a lysis time for a fibrin clot.The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Time for Lysis of Fibrin Clot                                                            Thrombin (unit/ml)                                                 Prourokinase 0.5          1.0   2.0                                           ______________________________________                                                   (Min)                                                              Q-RPK        53           43    37                                            Q-RPR        36           30    24                                            Q-QPR        37           30    25                                            Q (135)      54           63    95                                            Natural      55           65    92                                            Prourokinase                                                                  ______________________________________                                    

Table 2 shows that the present mutant prourokinases Q-RPK, Q-RPR, andQ-QPR rapidly lyse a fibrin clot in the presence of thrombin, incontrast to a natural type prourokinase and a mutant prourokinase Q(135)wherein the higher the concentration of thrombin, the longer the timenecessary for the lysis of a fibrin clot. C.Y. Liu et al. shows that anactivity of thrombin developing when the thrombus is formed in plasmareaches up to 15 units/ml, and this value is maintained for severalminutes after a complete conversion of fibrinogen to fibrin (literature4). From the above, it is considered that the present prourokinase isproperly activated, at the beginning of thrombus formation, and exhibitsa rapid thrombolytic action.

The present mutant polypeptides Q-RPK, Q-RPR, Q-QPR and Q-SGR arecharacterized by being activated by a cleavage between the 158th and159th positions, and have an additional advantages in that they are notinactivated by a cleavage between the 156th and 157th positions.

Example 11 Purification of Mutant Prourokinase Q-RPK

The plasmid pMUT9Q-RPK obtained in Example 3 was used to transform E.coli JM103 by a conventional procedure. The transformant thus obtainedwas cultured in 1 l of L-broth at 37° C with shaking, and when anabsorbance at 600 nm reached about 0.4, 10 ml of 0.1 Misopropyl-β-D-thiogalactopyranoside was added, followed by culturing foran additional four hours. Next, cells were recovered by centrifugationand disrupted by ultrasonication in 50 ml of 50 mM Tris-HCl (pH 8.0)buffer containing 0.1 M sodium chloride, until an absorbance at 600 nmbecame not more than 10. This disruptant solution was centrifuged at 15Krpm for 30 minutes, and the supernatant was discarded. The precipitatewas suspended in 50 mM Tris-HCl (pH 8.0) buffer containing 4 M guanidinehydrochloride, and the suspension was allowed to stand at a roomtemperature for 90 minutes to dissolve the precipitate.

Next, to the mixture was added 1500 ml of 50 mM Tris-HCl (pH 8.0) buffercontaining 0.27 mM reduced glutathione and 1.33 mMethylenediaminitetraacetic acid, and the mixture was allowed to stand at25° C for 15 hours. The solid ammonium chloride was gradually dissolvedin the mixture to 25% saturation at 4° C, and the mixture thencentrifuged to eliminate any insoluble material. To the supernatant wasadded solid ammonium chloride to 50% concentration, to salt out adesired gene product (0-RPK), and the salting out product, was recoveredby centrifugation and then dissolved in a mM Tris-HCl (pH 8.0) buffercontaining ammonium sulfate of 7% saturation and 1 M guanidinehydrochloride. The solution was centrifuged to eliminate insolublematerial, and the supernatant was applied to a column (φ1.5 cm×30 cm) ofphenyl Sepharose CL-4B (Pharmacia LP Biochemicals) equilibrated with 50mM Tris-HCl (pH 8.0) buffer containing ammonium sulfate of 7% saturationand 1 M guanidine hydrochloride. After washing the column with theequilibrating buffer, the desired gene product (Q-RPK) was eluted fromthe column using 50 mM Tris-HCl (pH 8.0) containing 1 M guanidinehydrochloride. Next, the elute was applied to a column (φ1.0 cm×30 cm)of zinc chelate Sepharose 6B equilibrated with 50 mM Tris-HCl (pH 8.0)buffer containing 0.5 M sodium chloride, and after washing the columnwith 150 ml of the equilibrating buffer, the desired gene product(Q-RPK) was eluted with 50 mM sodium acetate buffer (pH 5.4) containing0.5 M sodium chloride. It was confirmed by SDS-polyacrylamide gelelectrophoresis that the elute contained only a single protein. Theresults are shown in FIG. 9.

Example 12 Purification of Mutant Prourokinase Q-RPR

Using the plasmid pMUT9Q-RPR obtained in Example 4, and using the sameprocedure as in Example 11, the mutant prourokinase Q-RPR was purified.The purified preparation was confirmed to be a single protein bySDS-polyacrylamide gel electrophoresis. The results are shown in FIG. 9.

Example 13 Properties of Purified Mutant Prourokinases Q-RPK and Q-RPR

(i) Activation by plasmin with elapse of time

To 95 μl of 50 mM Tris-HCl (pH 8.0) buffer containing 0.01% TritonX-100, and 5 IU of purified mutant prourokinase Q-RPK or Q-RPR obtainedin Example 11 or 12 was added 5 μl of plasmin aqueous solutioncontaining 3×10⁴ CU of plasmin, and reactions were carried out at 37° C.for different times. The reactions were terminated by adding 50 μl ofaqueous solution containing 0.1 μg of soybean trypsin inhibitor. Next,the activity of mutant prourokinase contained in 105 μl of this reactionmixture was measured by the method described in (iv). Note, as acontrol, natural type urokinase and a purified preparation of mutanturokinase Q(135) P(157) (Japanese Unexamined Patent Publication No.62-143686) (EP 0210279) were used. The results are shown in FIG. 10.

FIG. 10 shows that the mutant prourokinases Q-RPK and Q-RPR areactivated by plasmin in the same way as the mutant prourokinase Q(135)D(157)

(ii) Activation by Thrombin with Elapse of Time

To 95 μl of 50 mM Tris-HCl (pH 8.2) buffer containing 0.01 Triton X-100,and 4 IU of a purified preparation of mutant prourokinase Q-RPK orQ-RPR, was added 5 μl of aqueous solution containing 4×10⁴ NIH units ofthrombin, and reactions were carried out for different times. Next, theactivity of mutant prourokinase contained in 105 μl of this reactionmixture was measured by a method described in (iv). The reactions wereterminated by adding 5 μl of aqueous solution containing 1×10³ NIH unitsof hirudine. Note, as control samples, natural type prourokinase and apurified preparation of mutant prourokinase Q(135) D(157) were used. Theresults are shown in FIG. 11.

FIG. 11 shows that the mutant prourokinases Q-RPK and Q-RPR are rapidlyactivated by thrombin. This property is an absolutely new property notfound in natural type prourokinase and mutant prourokinase Q(135)D(157).

(iii) Development of Residual Activity of Thrombin-Treated Preparation

To 85 μl of 50 mM Tris-HCl (pH 8.0) buffer containing 0.01 % TritonX-100, and mutant prourokinase Q-RPK or Q-RPR obtained in Example 11 or12, 5 μl of an aqueous solution containing 4×10⁴ NIH units of thrombinwas added, and after reactions were carried out at 37° C. for differenttimes, the reactions were terminated by adding 5 μl of an aqueoussolution containing 1×10³ NIH units of hirudine. Next, to the reactionmixture 5 μl of an aqueous solution containing 0.015 CU of plasmin wasadded, and after reaction was carried out at 37° C. for 30 minutes, thereaction was terminated by adding 5 μl of an aqueous solution containing5 μg of soybean trypsin. Next, the activity of mutant prourokinasecontained in 105 μl of this reaction mixture was measured by the methoddescribed in (iv). Note, as control samples natural type prourokinaseand a purified preparation of mutant prourokinase Q(135) D(157) wereused. The results are shown in FIG. 12.

FIG. 12 shows that the mutant prourokinases Q-RPK and Q-RPR are, incontrast to natural type prourokinase and mutant prourokinase Q(135)D(157), little inactivated by thrombin.

(iv) Measurement of Activity of Activated Mutant Prourokinase bySynthetic Substrate S-2444

To 105 μl of the reaction mixtures obtained in (i), (ii) and (iii), 0.7μl of 50 mM Tris-HCl (pH 8.0) solution containing 0.2 mM S-2444 (DaiichiKagaku Yakuhin), 0.1 M sodium chloride and 0.01 % Triton X-100, andafter reaction was carried out at 37° C. for 30 minutes, the reactionwas terminated by adding 0.1 ml of acetic acid. Next, an increase of anabsorbance at 405 nm for 30 minutes was measured, and the activity forthe sample was calculated on the basis of a value for a urokinasestandard (Nippon Soda K.K.) (increase of absorbance at 405 nm for 30minutes is 0.0608 for one unit). The results are shown in FIGS. 10, 11,and 12. FIG. 10 shows that the mutant prourokinase Q-RPK and Q-RPR are,similar to natural type prourokinase and mutant prourokinaseQ(135)D(157), activated by plasmin. FIG. 11 shows that the mutantprourokinase Q-RPK and Q-RPR are rapidly activated by thrombin. Thisproperty is an absolutely new property not found in natural typeprourokinase and mutant prourokinase Q(135)D(157). FIG. 12 shows thatthe mutant prourokinase Q-RPK and Q-RPR are, in contrast to natural typeprourokinase and the mutant prourokinase Q(135) D(157), littleinactivated by thrombin.

Reference Example 1. (Construction of Starting Plasmid)

Starting with plasmid pKYU22 containing a cDNA coding for natural typehuman prourokinase, the structure of a 30 bp of the 5'-end portion ofthis naturally occurring cDNA was altered to permit the prourokinasegene to be efficiently expressed in E. coli under the SD sequence of thePseudomonas putida-derived C230 gene.

Escherichia coli X1766 (pKYU22) containing plasmid pKYU22 was depositedas FERM BP-968, as an international deposition.

The following three single-chain DNA oligomers comprising 29, 15, and 20nucleotides respectively were synthesized by the phosphotriester method:##STR4##

Next, 1 μg each of the synthetic DNA oligomers was heated for 2 minutesat 95° C, phosphorylated at the 5'-end with T4 polynucleotide kinase andpurified using a Sep Pak (C18) column (made by Waters). After drying,the purified material was dissolved in 50 μl of 20 mM Tris-HCL (pH 7.6)and 10 mM MgCl , and annealed by heating for 2 minutes at 95° C.,cooling slowly to room temperature, and then maintaining the solutionovernight at 12° C. to give the following double-stranded DNA: ##STR5##

On the one hand, 5 μg of DNA of plasmid _(p) KYU22 was digested twicewith restriction endonucleases Bo1II and AatII, and about 5.7 Kb DNA.fragment was recovered by electric elution, and on the other hand, 5 μgof DNA of the same plasmid _(p) KYU22 was twice digested withrestriction endonucleases PstI and BqlII and about 400 bp DNA fragmentwas obtained by electric elution method. This fragment was againdigested with restriction endonucleases TaqI and about 260 bp DNAfragment was recovered by electric elution. These two different DNAfragments were recovered and purified by phenol/chloroform extraction,and precipitation with 2 volumes of ethanol.

The two different DNA fragments and the aforesaid double-strandedsynthetic DNA oligomer were ligated together using T4DNA ligase andtransformed into E. coli χ1776. Then the transformants were screened bythe rapid isolation method by the alkali lysis procedure, and a cloneEscherichia coli χ1776/pKMUl carrying the plasmid pKMUl that contains amodified prourokinase gene was obtained. The clone E. coli χ1776/pKYUlhas been deposited with the Fermentation Research Institute, Agency ofScience and Technology as FERM P-8040.

Reference Example 2. Construction of Prourokinase Directly ExpressivePlasmid (pMUT4L)

Five μg of plasmid KMUl from Reference Example 1 was digested with 10units of restriction endonuclease AatII and the digest was isolatedafter treatment with calf intestinal phosphatase (CIP). On the otherhand, 5 μg of plasmid pTCMl was digested with 10 units of restrictionendonuclease AatII and about 500 bp DNA fragment was isolated by theelectric elution method. These two different DNA fragments were purifiedby repeated phenol/chloroform extraction and ethanol precipitation.

Both of these DNA fragments were joined together using T4DNA ligase andwere transformed into E. coli JM103. The transformants were screened bythe rapid isolation method using the alkali lysis procedure, and a clonecarrying plasmid pMUTlL in which tac promoter/ operator and C230SDsequence having the normal orientation with reference to theprourokinase gene was obtained.

Said plasmid pTCMl, which is a novel plasmid constructed by the presentinventors, contains an expression controlling region comprising tacpromoter/ operator, lac SD, C230SD sequences as well as the C230structural gene. E. coli JM103/pTCMl carrying said plasmid has beendeposited with the Fermentation Research Institute, Agency of Scienceand Technology as FERM BP-1990 as an international deposition.

In plasmid MUTlL the modified prourokinase gene of the present inventionis inserted at an appropriate site downstream of the expressioncontrolling region comprising tac promoter/operator, lac SD and C230SDsequences.

Next, 5 μg of plasmid pKK223-3 (Literatures 22, 23 and 24) was digestedwith 10 units of restriction endonuclease HindIII, and the digest wastreated with calf intestinal phosphatase (P.L. Biochemicals).

On the other hand, 1 μg of the resulting plasmid pMUTlL was digestedwith 4 units of restriction endonuclease DraI and the digestion fragmentand 1 μg of 5'-phosphorylated HindIII linker (dCAAGCTTG) were ligatedwith T4DNA ligase. Digestion was carried out using 12 units ofrestriction endonuclease HindIII, and the digest was dissolved in 0.15 MNaCl. The solution was extracted with an equal volume ofphenol/chloroform and the DNA was precipitated by the addition of 2volumes of ethanol. The precipitate was collected at 16,000 rpm and at4° C. and was dried.

The resultant pMUTlL digestion fragment and the HindIII digestionfragment of aforesaid pKK223-3 were ligated using T4 DNA ligase and weretransformed into E. coli JM103. The transformants were screened by thealkali lysis procedure and a clone, E. coli JM103/pMUT2L containingplasmid pMUT2L was obtained.

This plasmid not only has said expression control region upstream fromthe prourokinase gene but also has a transcription terminator (rrnB, T₁T₂) of the E. coli ribosomal gene derived from pKK223-3 downstreamtherefrom.

Note, Plasmid pKK223-3 is readily obtainable as it is marketed byPharmacia P-L Biochemicals.

Five μg of plasmid pMUT2L was digested with 10 units each of restrictionendonucleases SphI and Tthlll I, and after extraction withphenol/chloroform DNA was precipitated with ethanol. The DNA thusrecovered was blunt-ended using T4 polymerase in the presence of 0.1 mMdGTP, dCTP, dATP and TTP, and was recycled by T4 DNA ligase. The DNA wastransformed into E. coli JM103 and colonies were allowed to form on anLB agar medium containing 50 μg/ml of ampicillin. The transformants werescreened by the alkali lysis procedure (literature 14) and a clone, E.coli JM103/pMUT4L was obtained.

LITERATURE

1. Gurewich, V. et al.; J.Clin. Invest. 73, 1731 (1984).

2 Ichinose, A. et al. ; J. Biol. Chem. 261, 3486 (1986).

3. Kasai, S. et al. ; J. Biol. Chem. 260, 12382 (1985).

4. Liu, C.Y. et al. ; J. Biol. Chem. 254, 10421 (1979).

5. Kaminski, M. et al. ; J. Biol. Chem. 258, 10530 (1983).

6. Jui-Yoa Chang ; Eur. J. Biochem. 151, 217 (1985).

7. Japanese Patent Application No. 61-12984 (Japanese Unexamined PatentPublication No. 62-143686)

Referring to deposition of microorganism under Rule 13-2.

DEPOSITORY AUTHORITY: Fermentation Research Institute Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry

ADDRESS:1-3, Higashi 1-chome Tsukuba-shi Ibaraki-ken 305, Japan

DEPOSITION NUMBER AND DATE OF DEPOSITION:

1. FERM BP-1990 Aug. 17, 1984 (Transferred from FERM p-7779 on August 4,1988)

2. FERM P-8040 Jan. 11, 1985

3. FERM BP-969 Jan. 11, 1985 (Transferred from FERM P-8042 on January22, 1986)

4. FERM BP-970 Apr. 18, 1985 (Transferred from FERM P-8188 on January22, 1986)

5. FERM BP-971 Jul. 11, 1985 (Transferred from FERM P-8341 on January22, 1986)

DEPOSITORY AUTHORITY: Deutsche Sammulung von Microorganismen

ADDRESS: Grisebachstrasse 8, D-3400 Gottingen, Fedral Republic ofGermany

DEPOSITION NUMBER AND DATA OF DEPOSITION:

    ______________________________________                                        6. DSM 4186    July 22, 1987                                                  7. DSM 4187    July 22, 1987                                                  8. DSM 4188    July 22, 1987                                                  9. DSM 4189    July 22, 1987                                                  ______________________________________                                    

We claim:
 1. A human prourokinase-like polypeptide having the followingamino acid sequence:

    (Met)·Ser.sup.1.....X.sup.156 ·Y.sup.157 ·Z.sup.158--

wherein Met is an optionally present methionine; Ser¹ is the firstN-terminal serine; X¹⁵⁶ .19 Y¹⁵⁷ ·Z¹⁵⁸ is an amino acid sequence fromthe 156th amino acid to the 158th amino acid and is selected from thegroup consisting of Arg.Pro.Lys, Arg.Pro.Arg, and Cln,Pro.Arg; thedotted line represents an amino acid sequence from the second amino acidto the 155th amino acid of native human prourolinase wherein the 135thLys is optionally replaced by an amino acid selected from the groupconsisting of Asn and Gln,; and the solid line represents an amino acidsequence from the 159th amino acid to the C-terminal of the native humanprourokinase; or substantially the same amino acid sequence as theabove-mentioned amino acid sequence.
 2. A polypeptide according to claim1, wherein the amino acid sequence of the native human prourokinase isan amino acid sequence encoded by cDNA corresponding to mRNA derivedfrom human kidney.
 3. A pharmaceutical preparation comprising a humanprourokinase polypeptide having the following amino acid sequence:

    (Met)·Ser.sup.1.....X.sup.156 ·Y.sup.157 ·Z.sup.158 --

wherein Met is an optionally present methionine; Ser¹ is the firstN-terminal serine; X¹⁵⁶ ·Y¹⁵⁷ ·Z¹⁵⁸ is an amino acid sequence from the156th amino acid to the 158th amino acid and is selected from the groupconsisting of Arg.Pro.Lys, Arg.Pro.Arg, and Gln.Pro.Arg; the dotted linerepresents an amino acid sequence from the second amino acid to the155th amino acid of native human prourokinase wherein the 135th Lys isoptionally replaced by an amino acid selected from the group consistingof Asn and Gln,; and the solid line represents an amino acid sequencefrom the 159th amino acid to the C-terminal of the native humanprourokinase; or substantially the same amino acid sequence as theabove-mentioned amino acid sequence.
 4. Method for the prophylaxis ortreatment of thrombus formation, characterized by administering to apatient a human prourokinase-like polypeptide having the following aminoacid sequence:

    (Met) ·Ser.sup.1..... X.sup.156 ·Y.sup.157 ·Z.sup.158 --

wherein Met is an optionally present methionine; Ser¹ is the firstN-terminal serine; X¹⁵⁶ ·Y¹⁵⁷ ·Z¹⁵⁸ is an amino acid sequence from the156th amino acid to the 158th amino acid and is selected from the groupconsisting of Arg.Pro.Lys, Arg.Pro.Arg, and Gln.Pro.Arg; the dotted linerepresents an amino acid sequence from the second amino acid to the155th amino acid of native human prourokinase wherein the 135th Lys isoptionally replaced by an amino acid selected from the group consistingof Asn and Gln,; and the solid line represents an amino acid sequencefrom the 159th amino acid to the C-terminal of the native humanprourokinase; or substantially the same amino acid sequence as theabove-mentioned amino acid sequence.